Over the past two decades, the number of Vibrio parahaemolyticus infections worldwide has increased dramatically. In the United States (U.S.), V. parahaemolyticus has emerged as the leading cause of seafood- borne gastroenteritis. A majority of these illnesses have been attributed to the appearance and expansion of a pandemic serotype (O3:K6). However, in the Pacific Northwest (PNW) region of the U.S., this highly virulent serotype is very rarely associated with outbreaks (with only three reported cases), despite its alarming abundance in the environment. Although no explanation for this curious etiology has materialized we have previously shown that environmental O3:K6 from this region exhibited decreased virulence in zebrafish. Subsequent genome sequencing has revealed that environmental O3:K6 from the PNW carry a novel prophage (Vibrio prophage NW1) that has displaced seven genes: VP1884-VP1890. To better understand pandemic V. parahaemolyticus dynamics we propose to investigate the genetic mechanisms that underlie the unique etiology of this pathogen in the PNW. Our central hypothesis is that prophage NW1 depresses virulence through the displacement of 7 genes including the cold-shock transcriptional regulator cspA (VP1889), the virulence-associated vacB (VP1890), and the hypothetical protein VP1888. The recent dramatic increase in the number of V. parahaemolyticus infections, the continued expansion of the range and seasonal window of infections (owing to climate warming), and the increasing reliance of the global population on seafood as a major source of animal protein all provide strong rationale for this study. The long-term goal of our research is to understand how prophage NW1 affects the virulence of pandemic V. parahaemolyticus O3:K6. To accomplish this goal, the investigators will address three specific aims using a combination of traditional and cutting-edge approaches: 1) screen a large collection of environmental isolates, including O3:K6 isolates, to determine the prevalence of prophage NW1, 2) quantify the virulence of O3:K6 isolates carrying prophage NW1 by intraperitoneal challenge in zebrafish and 3) investigate the mechanisms underpinning virulence by quantifying virulence in wild-type and prophage mutants. Results are expected to answer open questions about virulence and evolution in a pandemic bacterial pathogen that poses a significant threat to human health and food security. This study aligns closely with the NIAID mandate to respond to emerging public health threats as well as the NIAID mission to better understand, treat and ultimately prevent infectious diseases. Further, this study aligns closely with the goals of the AREA program in that it will strengthen the research environment at a minority-serving institution and expose underrepresented students to meritorious research.